U.S. patent application number 12/678362 was filed with the patent office on 2010-07-29 for microorganism concentration process.
Invention is credited to Manjiri T. Kshirsagar, Tushar A. Kshirsagar, Thomas E. Wood.
Application Number | 20100190171 12/678362 |
Document ID | / |
Family ID | 40721579 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190171 |
Kind Code |
A1 |
Kshirsagar; Manjiri T. ; et
al. |
July 29, 2010 |
MICROORGANISM CONCENTRATION PROCESS
Abstract
A process for capturing or concentrating microorganisms for
detection or assay comprises (a) providing a concentration agent
that comprises an amorphous metal silicate and that has a surface
composition having a metal atom to silicon atom ratio of less than
or equal to about 0.5, as determined by X-ray photoelectron
spectroscopy (XPS); (b) providing a sample comprising at least one
microorganism strain; and (c) contacting the concentration agent
with the sample such that at least a portion of the at least one
microorganism strain is bound to or captured by the concentration
agent.
Inventors: |
Kshirsagar; Manjiri T.;
(Woodbury, MN) ; Kshirsagar; Tushar A.; (Woodbury,
MN) ; Wood; Thomas E.; (Stillwater, MN) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Family ID: |
40721579 |
Appl. No.: |
12/678362 |
Filed: |
October 2, 2008 |
PCT Filed: |
October 2, 2008 |
PCT NO: |
PCT/US08/78587 |
371 Date: |
March 16, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60977180 |
Oct 3, 2007 |
|
|
|
Current U.S.
Class: |
435/5 ; 435/29;
435/6.1; 435/6.17; 435/6.18 |
Current CPC
Class: |
C12Q 1/24 20130101; G01N
33/54313 20130101; C12N 1/02 20130101; G01N 33/553 20130101; C12N
11/14 20130101; C12Q 1/04 20130101; G01N 33/552 20130101 |
Class at
Publication: |
435/6 ;
435/29 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A process comprising (a) providing a concentration agent that
comprises an amorphous metal silicate and that has a surface
composition having a metal atom to silicon atom ratio of less than
or equal to 0.5, as determined by X-ray photoelectron spectroscopy
(XPS); (b) providing a sample comprising at least one microorganism
strain; and (c) contacting said concentration agent with said
sample such that at least a portion of said at least one
microorganism strain is bound to or captured by said concentration
agent.
2. The process of claim 1, wherein said process further comprises
detecting the presence of at least one bound microorganism
strain.
3. The process of claim 1, wherein said concentration agent is a
particulate concentration agent.
4. The process of claim 1, wherein said surface composition has a
metal atom to silicon atom ratio of less than or equal to 0.4.
5. The process of claim 1, wherein said surface composition is at
least 10 average atomic percent carbon.
6. The process of claim 1, wherein said concentration agent has a
negative zeta potential at a pH of 7.
7. The process of claim 1, wherein said metal is selected from
magnesium, calcium, zinc, aluminum, iron, titanium, and
combinations thereof.
8. The process of claim 7, wherein said metal is magnesium.
9. The process of claim 1, wherein said concentration agent
comprises an amorphous metal silicate in at least partially fused
particulate form.
10. The process of claim 9, wherein said amorphous metal silicate
is spheroidized.
11. The process of claim 10, wherein said concentration agent is
amorphous, spheroidized magnesium silicate.
12. The process of claim 1, wherein said sample is in the form of a
fluid.
13. The process of claim 1, wherein said microorganism strain is
selected from strains of bacteria, fungi, yeasts, protozoans,
viruses, bacterial endospores, and combinations thereof.
14. (canceled)
15. The process of claim 1, wherein said contacting is carried out
by mixing said concentration agent and said sample.
16. The process of claim 2, wherein said detecting is carried out
by a method selected from culture-based methods, microscopy and
other imaging methods, genetic detection methods, immunologic
detection methods, bioluminescence-based detection methods, and
combinations thereof.
17. The process of claim 1, wherein said process further comprises
segregating the resulting microorganism-bound concentration
agent.
18. The process of claim 17, wherein said segregating is effected
by a method selected from gravitational settling, centrifugation,
filtration, and combinations thereof.
19. (canceled)
20. The process of claim 17, wherein said process further comprises
separating the resulting segregated concentration agent from said
sample.
21. A process comprising (a) providing a concentration agent that
comprises amorphous, spheroidized magnesium silicate and that has a
surface composition having a metal atom to silicon atom ratio of
less than or equal to 0.5, as determined by X-ray photoelectron
spectroscopy (XPS); (b) providing a sample comprising at least one
strain of microorganism selected from bacteria, yeasts, viruses,
bacterial endospores, and combinations thereof and (c) contacting
said concentration agent with said sample such that at least a
portion of said at least one strain of microorganism is bound to or
captured by said concentration agent.
22. (canceled)
23. (canceled)
24. (canceled)
25. A kit comprising (a) a concentration agent that comprises an
amorphous metal silicate and that has a surface composition having
a metal atom to silicon atom ratio of less than or equal to 0.5, as
determined by X-ray photoelectron spectroscopy (XPS); (b) a testing
container; and (c) instructions for using said concentration agent
in carrying out the process of claim 1.
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
Description
STATEMENT OF PRIORITY
[0001] This application claims the priority of U.S. Provisional
Application No. 60/977,180 filed Oct. 3, 2007, the contents of
which are hereby incorporated by reference.
FIELD
[0002] This invention relates to processes for capturing or
concentrating microorganisms such that they remain viable for
detection or assay. In other aspects, this invention also relates
to diagnostic kits for use in carrying out such concentration
processes.
BACKGROUND
[0003] Food-borne illnesses and hospital-acquired infections
resulting from microorganism contamination are a concern in
numerous locations all over the world. Thus, it is often desirable
or necessary to assay for the presence of bacteria or other
microorganisms in various clinical, food, environmental, or other
samples, in order to determine the identity and/or the quantity of
the microorganisms present.
[0004] Bacterial DNA or bacterial RNA, for example, can be assayed
to assess the presence or absence of a particular bacterial species
even in the presence of other bacterial species. The ability to
detect the presence of a particular bacterium, however, depends, at
least in part, on the concentration of the bacterium in the sample
being analyzed. Bacterial samples can be plated or cultured to
increase the numbers of the bacteria in the sample to ensure an
adequate level for detection, but the culturing step often requires
substantial time and therefore can significantly delay the
assessment results.
[0005] Concentration of the bacteria in the sample can shorten the
culturing time or even eliminate the need for a culturing step.
Thus, methods have been developed to isolate (and thereby
concentrate) particular bacterial strains by using antibodies
specific to the strain (for example, in the form of antibody-coated
magnetic or non-magnetic particles). Such methods, however, have
tended to be expensive and still somewhat slower than desired for
at least some diagnostic applications.
[0006] Concentration methods that are not strain-specific have also
been used (for example, to obtain a more general assessment of the
microorganisms present in a sample). After concentration of a mixed
population of microorganisms, the presence of particular strains
can be determined, if desired, by using strain-specific probes.
[0007] Non-specific concentration or capture of microorganisms has
been achieved through methods based upon carbohydrate and lectin
protein interactions. Chitosan-coated supports have been used as
non-specific capture devices, and substances (for example,
carbohydrates, vitamins, iron-chelating compounds, and
siderophores) that serve as nutrients for microorganisms have also
been described as being useful as ligands to provide non-specific
capture of microorganisms.
[0008] Various inorganic materials (for example, hydroxyapatite and
metal hydroxides) have been used to non-specifically bind and
concentrate bacteria. Physical concentration methods (for example,
filtration, chromatography, centrifugation, and gravitational
settling) have also been utilized for non-specific capture, with
and/or without the use of inorganic binding agents. Such
non-specific concentration methods have varied in speed, cost (at
least some requiring expensive equipment, materials, and/or trained
technicians), sample requirements (for example, sample nature
and/or volume limitations), space requirements, ease of use (at
least some requiring complicated multi-step processes), suitability
for on-site use, and/or effectiveness.
SUMMARY
[0009] Thus, we recognize that there is an urgent need for
processes for rapidly detecting pathogenic microorganisms. Such
processes will preferably be not only rapid but also low in cost,
simple (involving no complex equipment or procedures), and/or
effective under a variety of conditions (for example, with varying
types of sample matrices, varying bacterial loads, and varying
sample volumes).
[0010] Briefly, in one aspect, this invention provides a process
for non-specifically concentrating the strains of microorganisms
(for example, strains of bacteria, fungi, yeasts, protozoans,
viruses (including both non-enveloped and enveloped viruses), and
bacterial endospores) present in a sample, such that the
microorganisms remain viable for the purpose of detection or assay
of one or more of the strains. The process comprises (a) providing
a concentration agent (preferably, a particulate concentration
agent) that comprises an amorphous metal silicate and that has a
surface composition having a metal atom to silicon atom ratio of
less than or equal to about 0.5, as determined by X-ray
photoelectron spectroscopy (XPS) (for example, a spheroidized
talc); (b) providing a sample (preferably, in the form of a fluid)
comprising at least one microorganism strain; and (c) contacting
(preferably, by mixing) the concentration agent with the sample
such that at least a portion of the at least one microorganism
strain is bound to or captured by the concentration agent.
Preferably, the process further comprises detecting the presence of
the at least one bound microorganism strain (for example, by
culture-based, microscopy/imaging, genetic, bioluminescence-based,
or immunologic detection methods) and/or segregating (preferably,
by gravitational settling) the resulting microorganism-bound
concentration agent. The process can optionally further comprise
separating the resulting segregated concentration agent from the
sample.
[0011] The process of the invention does not target a specific
microorganism strain. Rather, it has been discovered that certain
relatively inexpensive, inorganic materials can be surprisingly
effective in capturing a variety of microorganisms. Such materials
can be used to concentrate the microorganism strains present in a
sample (for example, a food sample) in a non-strain-specific
manner, so that one or more of the microorganism strains
(preferably, one or more strains of bacteria) can be more easily
and rapidly assayed.
[0012] The process of the invention is relatively simple and low in
cost (requiring no complex equipment or expensive strain-specific
materials) and can be relatively fast (preferred embodiments
capturing at least about 70 percent (more preferably, at least
about 80 percent; most preferably, at least about 90 percent) of
the microorganisms present in a sample in less than about 30
minutes, relative to a corresponding control sample without
concentration agent). In addition, the process can be effective
with a variety of microoganisms (including pathogens such as both
gram positive and gram negative bacteria) and with a variety of
samples (different sample matrices and, unlike at least some prior
art methods, even samples having low microorganism content and/or
large volumes). Thus, at least some embodiments of the process of
the invention can meet the above-cited urgent need for low-cost,
simple processes for rapidly detecting pathogenic microorganisms
under a variety of conditions.
[0013] In another aspect, the invention also provides a diagnostic
kit for use in carrying out the process of the invention, the kit
comprising (a) a concentration agent (preferably, a particulate
concentration agent) that comprises a metal silicate and that has a
surface composition having a metal atom to silicon atom ratio of
less than or equal to about 0.5, as determined by X-ray
photoelectron spectroscopy (XPS); (b) a testing container
(preferably, a sterile testing container); and (c) instructions for
using the concentration agent in carrying out the process of the
invention. Preferably, the diagnostic kit further comprises one or
more components selected from microorganism culture media, lysis
reagents, buffers, bioluminescence detection assay components,
genetic detection assay components, and combinations thereof.
DETAILED DESCRIPTION
Definitions
[0014] As used in this patent application:
"sample" means a substance or material that is collected (for
example, to be analyzed) by a non-cosmetic method (that is, by a
method other than by application and/or removal of a composition
comprising the above-described concentration agent to a human
body); "sample matrix" means the components of a sample other than
microorganisms; "detection" means the identification of at least a
component of a microorganism, which thereby determines that the
microorganism is present; "genetic detection" means the
identification of a component of genetic material such as DNA or
RNA that is derived from a target microorganism; "immunologic
detection" means the identification of an antigenic material such
as a protein or a proteoglycan that is derived from a target
microorganism; "microorganism" means any cell having genetic
material suitable for analysis or detection (including, for
example, bacteria, yeasts, viruses, and bacterial endospores);
"microorganism strain" means a particular type of microorganism
that is distinguishable through a detection method (for example,
microorganisms of different genera, of different species within a
genera, or of different isolates within a species); and "target
microorganism" means any microorganism that is desired to be
detected.
Concentration Agent
[0015] Concentration agents suitable for use in carrying out the
process of the invention include those that comprise a metal
silicate and that have a surface composition having a metal atom to
silicon atom ratio of less than or equal to about 0.5 (preferably,
less than or equal to about 0.4; more preferably, less than or
equal to about 0.3; most preferably, less than or equal to about
0.2), as determined by X-ray photoelectron spectroscopy (XPS).
Preferably, the surface composition also comprises at least about
10 average atomic percent carbon (more preferably, at least about
12 average atomic percent carbon; most preferably, at least about
14 average atomic percent carbon), as determined by X-ray
photoelectron spectroscopy (XPS). XPS is a technique that can
determine the elemental composition of the outermost approximately
3 to 10 nanometers (nm) of a sample surface and that is sensitive
to all elements in the periodic table except hydrogen and helium.
XPS is a quantitative technique with detection limits for most
elements in the 0.1 to 1 atomic percent concentration range.
Preferred surface composition assessment conditions for XPS can
include a take-off angle of 90 degrees measured with respect to the
sample surface with a solid angle of acceptance of .+-.10
degrees.
[0016] Concentration or capture using the above-described
concentration agents is generally not specific to any particular
strain, species, or type of microorganism and therefore provides
for the concentration of a general population of microorganisms in
a sample. Specific strains of microorganisms can then be detected
from among the captured microorganism population using any known
detection method with strain-specific probes. Thus, the
concentration agents can be used for the detection of microbial
contaminants or pathogens (particularly food-borne pathogens such
as bacteria) in clinical, food, environmental, or other
samples.
[0017] When dispersed or suspended in water systems, inorganic
materials such as metal silicates exhibit surface charges that are
characteristic of the material and the pH of the water system. The
potential across the material-water interface is called the "zeta
potential," which can be calculated from electrophoretic mobilities
(that is, from the rates at which the particles of material travel
between charged electrodes placed in the water system). The
concentration agents used in carrying out the process of the
invention have zeta potentials that are more negative than that of,
for example, a common metal silicate such as ordinary talc. Yet the
concentration agents are surprisingly more effective than talc in
concentrating microorganisms such as bacteria, the surfaces of
which generally tend to be negatively charged. Preferably, the
concentration agents have a negative zeta potential at a pH of
about 7 (more preferably, a Smoluchowski zeta potential in the
range of about -9 millivolts to about -25 millivolts at a pH of
about 7; even more preferably, a Smoluchowski zeta potential in the
range of about -10 millivolts to about -20 millivolts at a pH of
about 7; most preferably, a Smoluchowski zeta potential in the
range of about -11 millivolts to about -15 millivolts at a pH of
about 7).
[0018] Useful metal silicates include amorphous silicates of metals
such as magnesium, calcium, zinc, aluminum, iron, titanium, and the
like (preferably, magnesium, zinc, iron, and titanium; more
preferably, magnesium), and combinations thereof. Preferred are
amorphous metal silicates in at least partially fused particulate
form (more preferably, amorphous, spheroidized metal silicates;
most preferably, amorphous, spheroidized magnesium silicate). Metal
silicates are known and can be chemically synthesized by known
methods or obtained through the mining and processing of raw ores
that are naturally-occurring.
[0019] Amorphous, at least partially fused particulate forms of
metal silicates can be prepared by any of the known methods of
melting or softening relatively small feed particles (for example,
average particle sizes up to about 25 microns) under controlled
conditions to make generally ellipsoidal or spheroidal particles
(that is, particles having magnified two-dimensional images that
are generally rounded and free of sharp corners or edges, including
truly or substantially circular and elliptical shapes and any other
rounded or curved shapes). Such methods include atomization, fire
polishing, direct fusion, and the like. A preferred method is flame
fusion, in which at least partially fused, substantially glassy
particles are formed by direct fusion or fire polishing of solid
feed particles (for example, as in the method described in U.S.
Pat. No. 6,045,913 (Castle), the description of which is
incorporated herein by reference). Most preferably, such methods
can be utilized to produce amorphous, spheroidized metal silicates
by converting a substantial portion of irregularly-shaped feed
particles (for example, from about 15 to about 99 volume percent;
preferably, from about 50 to about 99 volume percent; more
preferably, from about 75 to about 99 volume percent; most
preferably, from about 90 to about 99 volume percent) to generally
ellipsoidal or spheroidal particles.
[0020] Some amorphous metal silicates are commercially available.
For example, amorphous, spheroidized magnesium silicate is
commercially available for use in cosmetic formulations (for
example, as 3M.TM. Cosmetic Microspheres CM-111, available from 3M
Company, St. Paul, Minn.).
[0021] In addition to amorphous metal silicates, the concentration
agents can further comprise other materials including oxides of
metals (for example, iron or titanium), crystalline metal
silicates, other crystalline materials, and the like, provided that
the concentration agents have the above-described surface
compositions. The concentration agents, however, preferably contain
essentially no crystalline silica.
[0022] In carrying out the process of the invention, the
concentration agents can be used in any form that is amenable to
sample contact and microorganism capture (for example, in
particulate form or applied to a support such as a dipstick, film,
filter, tube, well, plate, beads, membrane, or channel of a
microfluidic device, or the like). Preferably, the concentration
agents are used in particulate form, more preferably comprising
microparticles (preferably, microparticles having a particle size
in the range of about 1 micrometer (more preferably, about 2
micrometers) to about 100 micrometers (more preferably, about 50
micrometers; even more preferably, about 25 micrometers; most
preferably, about 15 micrometers; where any lower limit can be
paired with any upper limit of the range).
Sample
[0023] The process of the invention can be applied to a variety of
different types of samples, including, but not limited to, medical,
environmental, food, feed, clinical, and laboratory samples, and
combinations thereof. Medical or veterinary samples can include,
for example, cells, tissues, or fluids from a biological source
(for example, a human or an animal) that are to be assayed for
clinical diagnosis. Environmental samples can be, for example, from
a medical or veterinary facility, an industrial facility, soil, a
water source, a food preparation area (food contact and non-contact
areas), a laboratory, or an area that has been potentially
subjected to bioterrorism. Food processing, handling, and
preparation area samples are preferred, as these are often of
particular concern in regard to food supply contamination by
bacterial pathogens.
[0024] Samples obtained in the form of a liquid or in the form of a
dispersion or suspension of solid in liquid can be used directly,
or can be concentrated (for example, by centrifugation) or diluted
(for example, by the addition of a buffer (pH-controlled)
solution). Samples in the form of a solid or a semi-solid can be
used directly or can be extracted, if desired, by a method such as,
for example, washing or rinsing with, or suspending or dispersing
in, a fluid medium (for example, a buffer solution). Samples can be
taken from surfaces (for example, by swabbing or rinsing).
Preferably, the sample is a fluid (for example, a liquid, a gas, or
a dispersion or suspension of solid or liquid in liquid or
gas).
[0025] Examples of samples that can be used in carrying out the
process of the invention include foods (for example, fresh produce
or ready-to-eat lunch or "deli" meats), beverages (for example,
juices or carbonated beverages), potable water, and biological
fluids (for example, whole blood or a component thereof such as
plasma, a platelet-enriched blood fraction, a platelet concentrate,
or packed red blood cells; cell preparations (for example,
dispersed tissue, bone marrow aspirates, or vertebral body bone
marrow); cell suspensions; urine, saliva, and other body fluids;
bone marrow; lung fluid; cerebral fluid; wound exudate; wound
biopsy samples; ocular fluid; spinal fluid; and the like), as well
as lysed preparations, such as cell lysates, which can be formed
using known procedures such as the use of lysing buffers, and the
like. Preferred samples include foods, beverages, potable water,
biological fluids, and combinations thereof (with foods, beverages,
potable water, and combinations thereof being more preferred).
[0026] Sample volume can vary, depending upon the particular
application. For example, when the process of the invention is used
for a diagnostic or research application, the volume of the sample
can typically be in the microliter range (for example, 10 .mu.L or
greater). When the process is used for a food pathogen testing
assay or for potable water safety testing, the volume of the sample
can typically be in the milliliter to liter range (for example, 100
milliliters to 3 liters). In an industrial application, such as
bioprocessing or pharmaceutical formulation, the volume can be tens
of thousands of liters.
[0027] The process of the invention can isolate microorganisms from
a sample in a concentrated state and can also allow the isolation
of microorganisms from sample matrix components that can inhibit
detection procedures that are to be used. In all of these cases,
the process of the invention can be used in addition to, or in
replacement of, other methods of microorganism concentration. Thus,
optionally, cultures can be grown from samples either before or
after carrying out the process of the invention, if additional
concentration is desired.
Contacting
[0028] The process of the invention can be carried out by any of
various known or hereafter-developed methods of providing contact
between two materials. For example, the concentration agent can be
added to the sample, or the sample can be added to the
concentration agent. A dipstick coated with concentration agent can
be immersed in a sample solution, a sample solution can be poured
onto a film coated with concentration agent, a sample solution can
be poured into a tube or well coated with concentration agent, or a
sample solution can be passed through a filter (for example, a
woven or nonwoven filter) coated with concentration agent.
[0029] Preferably, however, the concentration agent and the sample
are combined (using any order of addition) in any of a variety of
containers (optionally but preferably, a capped, closed, or sealed
container; more preferably, a capped test tube, bottle, or jar).
Suitable containers for use in carrying out the process of the
invention will be determined by the particular sample and can vary
widely in size and nature. For example, the container can be small,
such as a 10 microliter container (for example, a test tube) or
larger, such as a 100 milliliter to 3 liter container (for example,
an Erlenmeyer flask or a polypropylene large-mouth bottle). The
container, the concentration agent, and any other apparatus or
additives that contact the sample directly can be sterilized (for
example, by controlled heat, ethylene oxide gas, or radiation)
prior to use, in order to reduce or prevent any contamination of
the sample that might cause detection errors. The amount of
concentration agent that is sufficient to capture or concentrate
the microorganisms of a particular sample for successful detection
will vary (depending upon, for example, the nature and form of the
concentration agent and sample volume) and can be readily
determined by one skilled in the art. For example, 10 milligrams of
concentration agent per milliliter of sample can be useful for some
applications.
[0030] If desired, contacting can be effected by passing a
particulate concentration agent at least once through a sample (for
example, by relying upon gravitational settling over a period of,
for example, about 10 minutes). Contact can be enhanced by mixing
(for example, by stirring, shaking, or use of a rocking platform)
such that the particles of concentration agent repeatedly pass or
settle through a substantial portion of the sample. For small
volumes on the order of microliters (typically less than 0.5
milliliter), mixing can be rapid such as by vortexing or
"nutation," for example as described in U.S. Pat. No. 5,238,812
(Coulter et al.), the description of which is incorporated herein
by reference. For larger volumes on the order of greater than or
equal to 0.5 milliliters (typically 0.5 milliliter to 3 liters),
mixing can be achieved by gently tumbling the particulate
concentration agent and the sample in an "end over end" fashion,
for example as described in U.S. Pat. No. 5,576,185 (Coulter et
al.), the description of which is incorporated herein by reference.
Such tumbling can be accomplished, for example, by means of a
device configured to hold a test tube or other type of reaction
vessel and to slowly rotate the test tube or vessel in an "end over
end" manner. Contacting can be carried out for a desired period
(for example, for sample volumes of about 100 milliliters or less,
up to about 60 minutes of contacting can be useful; preferably,
about 15 seconds to about 10 minutes or longer; more preferably,
about 15 seconds to about 5 minutes).
[0031] Thus, in carrying out the process of the invention, mixing
(for example, agitation, rocking, or stirring) and/or incubation
(for example, at ambient temperature) are optional but preferred,
in order to increase microorganism contact with the concentration
agent. A preferred contacting method includes both mixing (for
example, for about 15 seconds to about 5 minutes) and incubating
(for example, for about 3 minutes to about 30 minutes) a
microorganism-containing sample (preferably, a fluid) with
particulate concentration agent. If desired, one or more additives
(for example, lysis reagents, bioluminescence assay reagents,
nucleic acid capture reagents (for example, magnetic beads),
microbial growth media, buffers (for example, to moisten a solid
sample), microbial staining reagents, washing buffers (for example,
to wash away unbound material), elution agents (for example, serum
albumin), surfactants (for example, Triton.TM. X-100 nonionic
surfactant available from Union Carbide Chemicals and Plastics,
Houston, Tex.), mechanical abrasion/elution agents (for example,
glass beads), and the like) can be included in the combination of
concentration agent and sample.
[0032] If desired, the concentration agent (alone or in combination
with, for example, antimicrobial materials and/or with carrier
materials in the form of liquids (for example, water or oils),
solids (for example, fabrics, polymers, papers, or inorganic
solids), gels, creams, foams, or pastes) can be applied to or
rubbed against a non-porous or porous, solid,
microorganism-contaminated or microorganism-contaminatable material
or surface (for example, for use as a "cleaning" agent). Binders,
stabilizers, surfactants, or other property modifiers can be
utilized, if desired.
[0033] For such use, the concentration agent can be applied to
woven or nonwoven fabrics and can be applied to disposable surfaces
such as paper, tissues, cotton swabs, as well as to a variety of
absorbent and nonabsorbent materials. For example, the
concentration agent can be incorporated into cloth or paper carrier
materials for use as "cleaning" wipes. The concentration agent can
be applied (for example, in the form of wipes or pastes comprising
a carrier material) to solid surfaces, for example, in home,
day-care, industrial, and hospital settings, for cleansing toys,
equipment, medical devices, work surfaces, and the like. When used
for cleansing or other purposes, the sample can be simultaneously
collected and contacted with the concentration agent in a single
step, if desired.
Segregation and/or Separation
[0034] Optionally but preferably, the process of the invention
further comprises segregation of the resulting microorganism-bound
concentration agent. Such segregation preferably can be achieved by
relying, at least in part, upon gravitational settling (gravity
sedimentation; for example, over a time period of about 5 minutes
to about 30 minutes). In some cases, however, it can be desirable
to accelerate segregation (for example, by centrifugation or
filtration) or to use combinations of any of the segregation
methods.
[0035] The process of the invention can optionally further comprise
separating the resulting microorganism-bound concentration agent
and the sample. For fluid samples, this can involve removal or
separation of the supernatant that results upon segregation.
Separation of the supernatant can be carried out by numerous
methods that are well-known in the art (for example, by decanting
or siphoning, so as to leave the microorganism-bound concentration
agent at the bottom of the container or vessel utilized in carrying
out the process).
[0036] The process of the invention can be carried out manually
(for example, in a batch-wise manner) or can be automated (for
example, to enable continuous or semi-continuous processing).
Detection
[0037] A variety of microorganisms can be concentrated and,
optionally but preferably, detected by using the process of the
invention, including, for example, bacteria, fungi, yeasts,
protozoans, viruses (including both non-enveloped and enveloped
viruses), bacterial endospores (for example, Bacillus (including
Bacillus anthracis, Bacillus cereus, and Bacillus subtilis) and
Clostridium (including Clostridium botulinum, Clostridium
difficile, and Clostridium perfringens)), and the like, and
combinations thereof (preferably, bacteria, yeasts, viruses,
bacterial endospores, fungi, and combinations thereof; more
preferably, bacteria, yeasts, viruses, bacterial endospores, and
combinations thereof; even more preferably, bacteria, viruses,
bacterials endospores, and combinations thereof; most preferably,
gram-negative bacteria, gram-positive bacteria, non-enveloped
viruses (for example, norovirus, poliovirus, hepatitis A virus,
rhinovirus, and combinations thereof), bacterial endospores, and
combinations thereof). The process has utility in the detection of
pathogens, which can be important for food safety or for medical,
environmental, or anti-terrorism reasons. The process can be
particularly useful in the detection of pathogenic bacteria (for
example, both gram negative and gram positive bacteria), as well as
various yeasts, molds, and mycoplasmas (and combinations of any of
these).
[0038] Genera of target microorganisms to be detected include, but
are not limited to, Listeria, Escherichia, Salmonella,
Campylobacter, Clostridium, Helicobacter, Mycobacterium,
Staphylococcus, Shigella, Enterococcus, Bacillus, Neisseria,
Shigella, Streptococcus, Vibrio, Yersinia, Bordetella, Borrelia,
Pseudomonas, Saccharomyces, Candida, and the like, and combinations
thereof. Samples can contain a plurality of microorganism strains,
and any one strain can be detected independently of any other
strain. Specific microorganism strains that can be targets for
detection include Escherichia coli, Yersinia enterocolitica,
Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio
parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes,
Staphylococcus aureus, Salmonella enterica, Saccharomyces
cerevisiae, Candida albicans, Staphylococcal enterotoxin ssp,
Bacillus cereus, Bacillus anthracis, Bacillus atrophaeus, Bacillus
subtilis, Clostridium perfringens, Clostridium botulinum,
Clostridium difficile, Enterobacter sakazakii, Pseudomonas
aeruginosa, and the like, and combinations thereof (preferably,
Staphylococcus aureus, Salmonella enterica, Saccharomyces
cerevisiae, Bacillus atrophaeus, Bacillus subtilis, Escherichia
coli, human-infecting non-enveloped enteric viruses for which
Escherichia coli bacteriophage is a surrogate, and combinations
thereof).
[0039] Microorganisms that have been captured or bound (for
example, by adsorption) by the concentration agent can be detected
by essentially any desired method that is currently known or
hereafter developed. Such methods include, for example,
culture-based methods (which can be preferred when time permits),
microscopy (for example, using a transmitted light microscope or an
epifluorescence microscope, which can be used for visualizing
microorganisms tagged with fluorescent dyes) and other imaging
methods, immunological detection methods, and genetic detection
methods. The detection process following microorganism capture
optionally can include washing to remove sample matrix
components.
[0040] Immunological detection is detection of an antigenic
material derived from a target organism, which is commonly a
biological molecule (for example, a protein or proteoglycan) acting
as a marker on the surface of bacteria or viral particles.
Detection of the antigenic material typically can be by an
antibody, a polypeptide selected from a process such as phage
display, or an aptamer from a screening process.
[0041] Immunological detection methods are well-known and include,
for example, immunoprecipitation and enzyme-linked immunosorbent
assay (ELISA). Antibody binding can be detected in a variety of
ways (for example, by labeling either a primary or a secondary
antibody with a fluorescent dye, with a quantum dot, or with an
enzyme that can produce chemiluminescence or a colored substrate,
and using either a plate reader or a lateral flow device).
[0042] Detection can also be carried out by genetic assay (for
example, by nucleic acid hybridization or primer directed
amplification), which is often a preferred method. The captured or
bound microorganisms can be lysed to render their genetic material
available for assay. Lysis methods are well-known and include, for
example, treatments such as sonication, osmotic shock, high
temperature treatment (for example, from about 50.degree. C. to
about 100.degree. C.), and incubation with an enzyme such as
lysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase
E, and viral enolysins.
[0043] Many commonly-used genetic detection assays detect the
nucleic acids of a specific microorganism, including the DNA and/or
RNA. The stringency of conditions used in a genetic detection
method correlates with the level of variation in nucleic acid
sequence that is detected. Highly stringent conditions of salt
concentration and temperature can limit the detection to the exact
nucleic acid sequence of the target. Thus microorganism strains
with small variations in a target nucleic acid sequence can be
distinguished using a highly stringent genetic assay. Genetic
detection can be based on nucleic acid hybridization where a
single-stranded nucleic acid probe is hybridized to the denatured
nucleic acids of the microorganism such that a double-stranded
nucleic acid is produced, including the probe strand. One skilled
in the art will be familiar with probe labels, such as radioactive,
fluorescent, and chemiluminescent labels, for detecting the hybrid
following gel electrophoresis, capillary electrophoresis, or other
separation method.
[0044] Particularly useful genetic detection methods are based on
primer directed nucleic acid amplification. Primer directed nucleic
acid amplification methods include, for example, thermal cycling
methods (for example, polymerase chain reaction (PCR), reverse
transcriptase polymerase chain reaction (RT-PCR), and ligase chain
reaction (LCR)), as well as isothermal methods and strand
displacement amplification (SDA) (and combinations thereof;
preferably, PCR or RT-PCR). Methods for detection of the amplified
product are not limited and include, for example, gel
electrophoresis separation and ethidium bromide staining, as well
as detection of an incorporated fluorescent label or radio label in
the product. Methods that do not require a separation step prior to
detection of the amplified product can also be used (for example,
real-time PCR or homogeneous detection).
[0045] Bioluminescence detection methods are well-known and
include, for example, adensosine triphosphate (ATP) detection
methods including those described in U.S. Pat. No. 7,422,868 (Fan
et al.), the descriptions of which are incorporated herein by
reference.
[0046] Since the process of the invention is non-strain specific,
it provides a general capture system that allows for multiple
microorganism strains to be targeted for assay in the same sample.
For example, in assaying for contamination of food samples, it can
be desired to test for Listeria monocytogenes, Escherichia coli,
and Salmonella all in the same sample. A single capture step can
then be followed by, for example, PCR or RT-PCR assays using
specific primers to amplify different nucleic acid sequences from
each of these microorganism strains. Thus, the need for separate
sample handling and preparation procedures for each strain can be
avoided.
Diagnostic Kit
[0047] A diagnostic kit for use in carrying out the process of the
invention comprises (a) an above-described concentration agent
(preferably, particulate); (b) a testing container (preferably, a
sterile testing container); and (c) instructions for using the
concentration agent in carrying out the process of the invention.
Preferably, the diagnostic kit further comprises one or more
components selected from microorganism culture or growth media,
lysis reagents, buffers, bioluminescence detection assay components
(for example, luminometer, lysis reagents, luciferase enzyme,
enzyme substrate, reaction buffers, and the like), genetic
detection assay components, and combinations thereof. A preferred
lysis reagent is a lytic enzyme supplied in a buffer, and preferred
genetic detection assay components include one or more primers
specific for a target microorganism.
[0048] For example, a preferred embodiment of the diagnostic kit of
the invention contains a particulate concentration agent (for
example, in a sterile disposable container such as a glass or
polypropylene vial), in combination with instructions for using
said agent in carrying out the process of the invention (for
example, by mixing the concentration agent with a fluid sample to
be analyzed, allowing the concentration agent to settle by gravity,
removing the resulting supernatant, and detecting the presence of
at least one concentration agent-bound target microorganism
strain). The concentration agent optionally can be hydrated in a
small volume of buffer with preservative to improve stability
during storage and transportation and/or can be
contained/aliquotted in a tear-open, sealed pouch to prevent
contamination. The concentration agent can be in the form of a
dispersion or suspension in a liquid or can be in powder form.
Preferably, the diagnostic kit comprises pre-measured aliquots (for
example, based upon sample volume) of particulate concentration
agent (more preferably, contained in one or more tear-open, sealed
pouches).
EXAMPLES
[0049] Objects and advantages of this invention are further
illustrated by the following examples, but the particular materials
and amounts thereof recited in these examples, as well as other
conditions and details, should not be construed to unduly limit
this invention.
Materials
[0050] Crystalline magnesium silicate concentration agent
(hereinafter, Talc) was purchased from Mallinckrodt Baker, Inc.
(Phillipsburg, N.J.). All microorganism cultures were purchased
from The American Type Culture Collection (ATCC; Manassas,
Va.).
[0051] Amorphous, spheroidized magnesium silicate concentration
agent (hereinafter, AS-Talc) was obtained as 3M.TM. Cosmetic
Microspheres CM-111 (shaped as solid spheres; particle density of
2.3 g/cubic centimeter; surface area of 3.3 m.sup.2/g; particle
size: 90 percent less than about 11 microns, 50 percent less than
about 5 microns, 10 percent less than about 2 microns; available
from 3M Company, St. Paul, Minn.).
Zeta Potential Measurements
[0052] Zeta potentials of aqueous dispersions of the Talc and
AS-Talc concentration agents (5.75 weight percent Talc and 5.8
weight percent AS-Talc, respectively, in 18 mega ohms deionized
water obtained by using a Milli-Q.TM. Elix 10.TM. Synthesis A10
deionization system from Millipore Corporation, Bedford, Mass.)
were measured as a function of added hydrochloric acid (pH) using a
Colloidal Dynamics Acoustosizer II.TM. multi-frequency
electroacoustic spectral analyzer (Colloidal Dynamics, Warwick,
R.I.) equipped with a TM200 automatic titration module, pH
electrode, and in-line conductivity cell. Measurements were made
using polar calibration and polar sample settings with the
following general parameters:
TABLE-US-00001 Starting Volume: 170 mL of dispersion Titration
Volume: 5 to 10 mL at finish; 20 steps for each titration Titrant:
1.0 N hydrochloric acid in water (J. T. Baker, Phillipsburg, NJ)
Stir Rate: 300 revolutions per minute (rpm) Pump Rate: 400 mL per
minute Mixing Delay: 120 seconds with stirring after acid addition
before measurement
[0053] At a pH of about 7, the AS-Talc exhibited a Smoluchowski
zeta potential of about -12 mV, and the Talc exhibited a
Smoluchowski zeta potential of about -8 mV.
Surface Composition Analysis
[0054] The surface compositions of samples of the Talc and AS-Talc
concentration agents were analyzed by X-ray photoelectron
spectroscopy (XPS; also known as ESCA). Samples of the powders were
pressed onto double-sided, pressure sensitive adhesive tapes on
aluminum foil. Excess powder was removed from each sample surface
by blowing with compressed nitrogen gas.
[0055] Spectral data was acquired using a Kratos AXIS Ultra.TM. DLD
spectrometer (Kratos Analytical, Manchester, England) having a
monochromatic Al--K.sub..alpha. X-ray excitation source (1487 eV)
and a hemispherical electron energy analyzer operated in a constant
pass energy mode. The emitted photoelectrons were detected at a
take-off angle of 90 degrees measured with respect to the sample
surface with a solid angle of acceptance of .+-.10 degrees. A
low-energy electron flood gun was used to minimize surface
charging. Measurements were made using a 140 Watt power to anode
and 2.times.10.sup.-8 Torr chamber pressure.
[0056] An area of the surface of each concentration agent sample
measuring about 300 micrometers by about 700 micrometers was
analyzed for each data point. Three areas on each sample were
analyzed and averaged to obtain the reported average atomic percent
values. Data processing was carried out using standard Vision2.TM.
software (Kratos Analytical, Manchester, England). Results
(elements present at a detectable level by XPS on the surface of
the concentration agents) are shown in Table A below:
TABLE-US-00002 TABLE A Magnesium Silicon Carbon Oxygen Concen-
(Average (Average Ratio of (Average (Average tration Atomic Atomic
Magnesium Atomic Atomic Agent Percent) Percent) to Silicon Percent)
Percent) Talc 17 26 0.65 6.9 50 AS-Talc 6.5 32 0.20 14 47
Microorganism Concentration Test Method
[0057] An isolated microorganism colony was inoculated into 5 mL
BBL.TM. Trypticase.TM. Soy Broth (Becton Dickinson, Sparks, Md.)
and incubated at 37.degree. C. for 18-20 hours. This overnight
culture at .about.10.sup.9 colony forming units per mL was diluted
in adsorption buffer (containing 5 mM KCl, 1 mM CaCl.sub.2, 0.1 mM
MgCl.sub.2, and 1 mM K.sub.2HPO.sub.4) at pH 7.2 to obtain 10.sup.3
microorganisms per mL dilution. A 1.1 mL volume of the
microorganism dilution was added to separate, labeled sterile 5 mL
polypropylene tubes (BD Falcon.TM., Becton Dickinson, Franklin
Lakes, N.J.) containing 10 mg of concentration agent, each of which
was capped and mixed on a Thermolyne Maximix Plus.TM. vortex mixer
(Barnstead International, Iowa). Each capped tube was incubated at
room temperature (25.degree. C.) for 15 minutes on a Thermolyne
Vari Mix.TM. shaker platform (Barnstead International, Iowa). After
the incubation, each tube was allowed to stand on the lab bench for
10 minutes to settle the concentration agent. Control sample tubes
containing 1.1 mL of the microorganism dilution without
concentration agent were treated in the same manner. The resulting
settled concentration agent and/or supernatant (and the control
samples) were then used for analysis.
[0058] The settled concentration agent was re-suspended in 1 mL
sterile Butterfield's Buffer solution (pH 7.2.+-.0.2; monobasic
potassium phosphate buffer solution; VWR Catalog Number 83008-093,
VWR, West Chester, Pa.) and plated on 3M.TM. Petrifilm.TM. Aerobic
Count Plates culture medium (dry, rehydratable; 3M Company, St.
Paul., MN) according to the manufacturer's instructions. Aerobic
count was quantified using a 3M.TM. Petrifilm.TM. Plate Reader (3M
Company, St. Paul., MN). Results were calculated using the
following formula:
Percent CFU/mL in Re-suspended Concentration Agent=(number of
colonies from plated re-suspended concentration agent)/(number of
colonies from plated untreated control sample).times.100
(where CFU=Colony Forming Unit, which is a unit of live or viable
microorganisms). Results were then reported in terms of percent
capture of microorganisms by the concentration agent using the
formula below:
Capture Efficiency or Percent Capture=Percent CFU/mL in
Re-suspended Concentration Agent
[0059] For comparison purposes, in at least some cases 1 mL of the
supernatant was removed and plated undiluted or diluted 1:10 in
Butterfield's Buffer solution and plated onto 3M.TM. Petrifilm.TM.
Aerobic Count Plates culture medium. Aerobic count was quantified
using a 3M.TM. Petrifilm.TM. Plate Reader (3M Company, St. Paul.,
MN). Results were calculated using the following formula:
Percent CFU/mL in Supernatant=(number of colonies from plated
supernatant)/(number of colonies from plated untreated control
sample).times.100
(where CFU=Colony Forming Unit, which is a unit of live or viable
microorganisms). When the microorganism colonies and the
concentration agent were similar in color (providing little
contrast for the plate reader), results were based upon the
supernatant and were then reported in terms of percent capture of
microorganisms by the concentration agent using the formula
below:
Capture Efficiency or Percent Capture=100-Percent CFU/mL in
Supernatant
Examples 1 and 2 and Comparative Examples 1 and 2
[0060] Using the above-described microorganism concentration test
method, 10 mg amorphous, spheroidized magnesium silicate (prepared
as described above; hereinafter, AS-Talc) and crystalline
(non-spheroidized) magnesium silicate (hereinafter, Talc) were
tested separately for bacterial concentration against target
microorganisms, the gram-negative bacterium Salmonella enterica
subsp.enterica serovar Typhimurium (ATCC 35987) and the
gram-positive bacterium Staphylococcus aureus (ATCC 6538). The
results are shown in Table 1 below (standard deviation for all
samples less than 10 percent).
TABLE-US-00003 TABLE 1 Concentration Percent Example No.
Microorganism Agent Capture C-1 Staphylococcus Talc 58 1
Staphylococcus AS-Talc 99 C-2 Salmonella Talc 69 2 Salmonella
AS-Talc 92
Examples 3-5 and Comparative Examples 3-5
[0061] Using the above-described microorganism concentration test
method, different weights per unit volume of AS-Talc and Talc were
tested separately for bacterial concentration of the target
microorganism, Salmonella enterica subsp.enterica serovar
Typhimurium (ATCC 35987). The results are shown in Table 2 below
(standard deviation for all samples less than 10 percent).
TABLE-US-00004 TABLE 2 Amount of Concentration Concentration Agent
Percent Example No. Microorganism Agent (mg/mL) Capture C-3
Salmonella Talc 1 63 3 Salmonella AS-Talc 1 82 C-4 Salmonella Talc
5 64 4 Salmonella AS-Talc 5 90 C-5 Salmonella Talc 10 69 5
Salmonella AS-Talc 10 95
Examples 6-8 and Comparative Examples 6-8
[0062] Using the above-described microorganism concentration test
method, 10 mg of AS-Talc and Talc were tested separately against
different bacterial concentrations of the target microorganism,
Salmonella enterica subsp.enterica serovar Typhimurium (ATCC
35987). The results are shown in Table 3 below.
TABLE-US-00005 TABLE 3 Percent Microorganism Capture .+-. Example
Concentration Concentration Standard No. Microorganism Agent
(CFU/mL) Deviation C-6 Salmonella Talc 10 68 .+-. 9 6 Salmonella
AS-Talc 10 92 .+-. 11 C-7 Salmonella Talc 100 74 .+-. 3 7
Salmonella AS-Talc 100 98 .+-. 3 C-8 Salmonella Talc 1000 69 .+-. 1
8 Salmonella AS-Talc 1000 92 .+-. 1
Examples 9-11 and Comparative Examples 9-11
[0063] Using the above-described microorganism concentration test
method, 10 mg of AS-Talc and Talc were tested separately for
bacterial concentration of the target microorganism, Salmonella
enterica subsp.enterica serovar Typhimurium (ATCC 35987) for 5, 10,
and 15 minutes of incubation. The results are shown in Table 4
below (standard deviation for all samples less than 10
percent).
TABLE-US-00006 TABLE 4 Incubation Concentration Time Percent
Example No. Microorganism Agent (minutes) Capture C-9 Salmonella
Talc 5 74 9 Salmonella AS-Talc 5 97 C-10 Salmonella Talc 10 77 10
Salmonella AS-Talc 10 96 C-11 Salmonella Talc 15 75 11 Salmonella
AS-Talc 15 92
Example 12 and Comparative Example 12
[0064] Using the above-described microorganism concentration test
method, with the exception of the use of Butterfield's Buffer
solution instead of adsorption buffer, 10 mg of AS-Talc and Talc
were tested separately for yeast concentration of the target
microorganism, Saccharomyces cerevisiae (10.sup.2 CFU/mL; ATCC
201390). The resulting materials were plated on 3M.TM.
Petrifilm.TM. Yeast and Mold Count Plate culture medium (dry,
rehydratable; 3M Company, St. Paul, Minn.) and incubated for 5 days
according to the manufacturer's instructions. Isolated yeast
colonies were counted manually, and percent capture was calculated
as described above. Percent capture was 97 percent for AS-Talc and
82 percent for Talc (standard deviation for all samples less than
10 percent).
Examples 13-15
[0065] Food samples were purchased from a local grocery store (Cub
Foods, St. Paul). Turkey slices and apple juice (11 g) were weighed
in sterile glass dishes and added to sterile Stomacher.TM.
polyethylene filter bags (Seward Corp, Norfolk, UK). The food
samples were spiked with bacterial cultures at a 10.sup.2 CFU/mL
concentration using an 18-20 hour overnight culture (stock) of
Salmonella enterica subsp.enterica serovar Typhimurium (ATCC
35987). This was followed by the addition of 99 mL of Butterfield's
Buffer solution to each spiked sample. The resulting samples were
blended for a 2-minute cycle in a Stomacher.TM. 400 Circulator
laboratory blender (Seward Corp. Norfolk, UK). The blended samples
were collected in sterile 50 mL centrifuge tubes (BD Falcon.TM.,
Becton Dickinson, Franklin Lakes, N.J.) and centrifuged at 2000
revolutions per minute (rpm) for 5 minutes to remove large debris.
The resulting supernatants were used as samples for further
testing. The pH of the apple juice-based supernatant was adjusted
to 7.2 before testing by adding 1N sodium hydroxide (VWR, West
Chester, Pa.). Potable water (100 mL) from a drinking fountain was
collected in a sterile 250 mL glass bottle (VWR, West Chester, Pa.)
and was inoculated with the target microorganism Salmonella
enterica subsp.enterica serovar Typhimurium (ATCC 35987) at
10.sup.2 CFU/mL, mixed manually end-to-end 5 times, and incubated
at room temperature (25.degree. C.) for 15 minutes. This water
sample was used for further testing.
[0066] Using the above-described microorganism concentration test
method, each 1 mL test sample prepared as above was added
separately to a test tube containing 10 mg of AS-Talc and tested
for bacterial concentration of the target microorganism, Salmonella
enterica subsp.enterica serovar Typhimurium (ATCC 35987). The
results are shown in Table 5 below (standard deviation for all
samples less than 10 percent).
TABLE-US-00007 TABLE 5 Concentration Percent Example No.
Microorganism Agent Sample Capture 13 Salmonella AS-Talc Apple
Juice 86 14 Salmonella AS-Talc Turkey 78 15 Salmonella AS-Talc
Potable 98 Water
Examples 16 and 17
[0067] AS-Talc was tested for concentration of the target
microorganism Salmonella enterica subsp.enterica serovar
Typhimurium (ATCC 35987) from large-volume samples (300 mg AS-Talc
per 30 mL sample volume). Potable water (100 mL) from a drinking
fountain was collected in a sterile 250 mL glass bottle (VWR, West
Chester, Pa.) and inoculated with the target microorganism
Salmonella enterica subsp. enterica serovar Typhimurium (ATCC
35987) at 10.sup.2 CFU/mL. The resulting inoculated water was mixed
manually end-to-end 5 times and incubated at room temperature
(25.degree. C.) for 15 minutes. 30 mL samples of the incubated
water were added to sterile 50 mL conical polypropylene centrifuge
tubes (BD Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.)
containing 300 mg of AS-Talc and were tested by using the
above-described microorganism concentration test method. The
resulting settled AS-Talc was re-suspended in 30 mL sterile
Butterfield's Buffer solution, and 1 mL of the resulting suspension
was plated on 3M.TM. Petrifilm.TM. Aerobic Count Plates culture
medium. Percent capture was 98 percent (standard deviation less
than 10 percent).
[0068] Whole grape tomatoes (11 g) from a local grocery store (Cub
Foods, St. Paul) were placed in a sterile petridish and were
inoculated with the target microorganism Salmonella enterica
subsp.enterica serovar Typhimurium (ATCC 35987) at 10.sup.2 CFU/mL,
mixed manually by swirling 5 times, and incubated at room
temperature (25.degree. C.) for 5 minutes. The tomatoes were added
to sterile Stomacher.TM. polyethylene filter bags (Seward Corp,
Norfolk, UK) containing 99 mL of Butterfield's Buffer solution. The
contents of the bags were mixed by swirling for 1 minute. 30 mL
samples were added to sterile 50 mL conical polypropylene
centrifuge tubes (BD Falcon.TM., Becton Dickinson, Franklin Lakes,
N.J.) containing 300 mg of AS-Talc and tested for bacterial
concentration using the above-described microorganism concentration
test method. The AS-Talc particles were settled by centrifugation
at 2000 rpm for 5 minutes (Eppendorf, Westbury, N.Y.). The settled
particles were re-suspended in 30 mL sterile Butterfield's Buffer
solution, and 1 mL of the resulting suspension was plated on 3M.TM.
Petrifilm.TM. Aerobic Count Plates culture medium. Percent capture
was 99 percent (standard deviation less than 10 percent).
Examples 18 and 19
[0069] 10 mg of AS-Talc was tested for concentration of the target
bacterial endospores Bacillus atrophaeus (ATCC 9372) and Bacillus
subtilis (ATCC 19659). The above-described microorganism
concentration test method was utilized with the following
modifications: the overnight cultures had 2.times.10.sup.2 CFU/mL
Bacillus atrophaeus and 7.times.10.sup.2 CFU/mL Bacillus subtilis,
respectively; the resulting supernatants were plated undiluted; the
settled concentration agent with bound Bacillus atrophaeus was
resuspended in 1 mL sterile Butterfield's Buffer solution and
plated; and the settled concentration agent with bound Bacillus
subtilis was resuspended in 5 mL sterile Butterfield's Buffer
solution and plated (1 mL each). Capture efficiencies were
calculated based on counts from the plated supernatants, and the
results are shown in Table 6 below (standard deviation for all
samples less than 10 percent).
TABLE-US-00008 TABLE 6 Example Concentration Percent No.
Microorganism Agent Capture 18 Bacillus atrophaeus AS-Talc 97 19
Bacillus subtilis AS-Talc 95
Examples 20 and 21
[0070] 10 mg of AS-Talc was tested for concentration of the target
non-enveloped, bacteria-infecting virus, Escherichia coli
bacteriophage MS2 (ATCC 15597-B1; which is often used as a
surrogate for various human-infecting, non-enveloped enteric
viruses). A double layer agar method (described below) was used to
assay for capture of the Escherichia coli bacteriophage MS2 (ATCC
15597-B1) using Escherichia coli bacteria (ATCC 15597) as host.
[0071] Escherichia coli bacteriophage MS2 stock was diluted
ten-fold serially in sterile 1.times. adsorption buffer (containing
5 mM KCl, 1 mM CaCl.sub.2, 0.1 mM MgCl.sub.2, and 1 mM
K.sub.2HPO.sub.4) at pH 7.2 to obtain two dilutions with 10.sup.3
and 10.sup.2 plaque forming units per milliliter (PFUs/mL),
respectively. A 1.0 mL volume of resulting bacteriophage dilution
was added to a labeled sterile 5 mL polypropylene tube (BD
Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) containing 10
mg of concentration agent and mixed on a Thermolyne Maximix
Plus.TM. vortex mixer (Barnstead International, Iowa). The capped
tube was incubated at room temperature (25.degree. C.) for 15
minutes on a Thermolyne Vari Mix.TM. shaker platform (Barnstead
International, Iowa). After the incubation, the tube was allowed to
stand on the lab bench for 10 minutes to settle the concentration
agent. A control sample tube containing 1.0 mL of the bacteriophage
dilution without concentration agent was treated in the same
manner. The resulting settled concentration agent and supernatant
(and the control sample) were then used for analysis.
[0072] 100 microliters of the supernatant was removed and assayed
for bacteriophage using the double layer agar method described
below. An additional 800 microliters of supernatant was removed and
discarded. One hundred microliters of the settled concentration
agent was also assayed for bacteriophage.
Double Layer Agar Method:
[0073] A single colony of Escherichia coli bacteria (ATCC 15597)
was inoculated into 25 mL sterile 3 weight percent tryptic soy
broth (Bacto.TM. Tryptic Soy Broth, Becton Dickinson and Company,
Sparks, Md.; prepared according to manufacturer's instructions) and
incubated at 37.degree. C. in a shaker incubator (Innova.TM.44, New
Brunswick Scientific Co., Inc., Edison, N.J.) set at 250
revolutions per minute (rpm) overnight. 750 microliters of this
overnight culture was used to inoculate 75 mL sterile 3 weight
percent tryptic soy broth. The resulting culture was incubated at
37.degree. C. in the shaker incubator set at 250 rpm to obtain
Escherichia coli cells in the exponential phase as measured by
absorbance at 550 nm (absorbance values 0.3-0.6) using a SpectraMax
M5 spectrophotometer (Molecular Devices, Sunnyvale, Calif.). The
cells were incubated on ice until used for assay.
[0074] One hundred microliters of the above-described bacteriophage
test samples were mixed with 75 microliters of the ice-incubated
Escherichia coli (host bacteria) cells and incubated at room
temperature (25.degree. C.) for 5 minutes. The resulting samples
were mixed with 5 mL sterile molten top agar (3 weight percent
tryptic soy broth, 1.5 weight percent NaCl, 0.6 weight percent
agar; prepared that day and maintained in a 48.degree. C.
waterbath). The mixture was then poured on top of bottom agar (3
weight percent tryptic soy broth, 1.5 weight percent NaCl, 1.2
weight percent agar) in petridishes. The molten agar component of
the mixture was allowed to solidify for 5 minutes, and the
petridishes or plates were inverted and incubated at 37.degree. C.
The plates were visually inspected after overnight incubation, and
those plates containing settled concentration agent (as well as the
control plate) showed the presence of bacteriophage plaques.
Capture efficiencies were calculated based on counts from the
plated supernatants and determined to be 72 percent for the
10.sup.2 PFU/mL dilution (standard deviation less than 10
percent).
Example 22
[0075] Apple juice was purchased from a local grocery store (Cub
Foods, St. Paul). Apple juice (11 g) was weighed in a sterile glass
dish and added to 99 mL sterile Butterfield's Buffer. The resulting
combination was mixed by swirling for 1 minute and was spiked with
two bacterial cultures, each at a 1 CFU/mL concentration, using
18-20 hour overnight cultures (bacterial stocks) of Salmonella
enterica subsp. enterica serovar Typhimurium (ATCC 35987) and
Escherichia coli (ATCC 51813). Serial dilutions of the bacterial
stocks had been made in 1.times. adsorption buffer as described
above.
[0076] Using the above-described microorganism concentration test
method, a 10 mL volume of the spiked apple juice sample was added
to a sterile 50 mL conical polypropylene centrifuge tube (BD
Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) containing 100
mg of AS-Talc and incubated for 15 minutes for bacterial
capture/concentration of the target microorganism, Salmonella (in
the presence of the Escherichia coli, a competitor microorganism).
The resulting supernatant was removed, and the settled
concentration agent was transferred to another sterile 50 mL tube
containing 2 mL sterile 3 weight percent tryptic soy broth
(Bacto.TM. Tryptic Soy Broth, Becton Dickinson and Company, Sparks,
Md.; prepared according to manufacturer's instructions). The tube
was loosely capped, and its contents were mixed and incubated at
37.degree. C. After overnight incubation, the resulting broth
mixture was tested for the presence of Salmonella using a
RapidChek.TM. Salmonella lateral flow immunoassay test strip from
SDI (Strategic Diagnostics, Inc., Newark, Del.). Visual inspection
of the test strip showed it to be positive for Salmonella.
[0077] Nucleic acid detection by polymerase chain reaction (PCR)
was also carried out for the microorganism-containing broth
mixture. 1 mL of the above-described overnight-incubated,
concentration agent-containing broth was assayed as a test sample
for the presence of Salmonella by using a TaqMan.TM. ABI Salmonella
enterica Detection Kit from Applied Biosystems (Foster City,
Calif.). As a control sample, 1 mL of the 18-20 hour overnight
culture (stock) of Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 35987) was also assayed. PCR testing was
conducted in a Stratagene Mx3005P.TM. QPCR (quantitative PCR)
System (Stratagene Corporation, La Jolla, Calif.) by using the
following cycle conditions per cycle for 45 cycles: 25.degree. C.
for 30 seconds, 95.degree. C. for 10 minutes, 95.degree. C. for 15
seconds, and 60.degree. C. for 1 minute. An average (n=2) cycle
threshold value (CT value) of 17.71 was obtained for the control
sample. An average (n=2) CT value of 19.88 was obtained for the
test sample containing concentration agent, indicating a positive
PCR reaction and confirming the presence of Salmonella.
[0078] The referenced descriptions contained in the patents, patent
documents, and publications cited herein are incorporated by
reference in their entirety as if each were individually
incorporated. Various unforeseeable modifications and alterations
to this invention will become apparent to those skilled in the art
without departing from the scope and spirit of this invention. It
should be understood that this invention is not intended to be
unduly limited by the illustrative embodiments and examples set
forth herein and that such examples and embodiments are presented
by way of example only, with the scope of the invention intended to
be limited only by the claims set forth herein as follows:
* * * * *